Quantitative and semi-quantitative measurements of axonal degeneration in tissue and primary neuron cultures

Axon viability is critical for maintaining neural connectivity, which is central to neural functionality. Many neurodegenerative diseases (e.g., Parkinson's disease (PD) and Alzheimer's disease) appear to involve extensive axonal degeneration that often precedes somatic loss in affected neural populations. Axonal degeneration involves a number of intracellular pathways and characteristic changes in axon morphology (i.e., swelling, fragmentation, and loss).

We describe a relatively simple set of methods to quantify the axonal degeneration using the 6-hydroxydopamine neurotoxin model of PD in rats and a colchicine-induced model in primary rat neurons. Specifically, approaches are described that use the spaceballs stereological probe for tissue sections and petrimetrics stereological probe for cultured neurons, and image analysis techniques in both tissue sections and cultured neurons.

These methods provide a mechanism for obtaining quantitative and semi-quantitative data to track the extent of axonal degeneration and may prove useful as outcome measures in studies aimed at preventing or slowing axonal degeneration in disease models.

Existing methods of quantification of axonal degeneration use densitometry and manual counts of axonal projections, but they do not utilize the random, unbiased systematic sampling approaches that are characteristic of stereological methods. The ImageJ thresholding analyses described here provide a descriptive method for quantifying the state of axonal degeneration.

These methods provide an efficient and effective means to quantify the extent and state of axonal degeneration in animal tissue and cultured neurons and can be used in other models for the same purposes.